RESUMEN
The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Cocultivo , Sinergismo Farmacológico , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos NOD , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/biosíntesis , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Nucleofosmina , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Distribución Aleatoria , Transcripción Genética/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/biosíntesis , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
During erythropoiesis, haematopoietic stem cells (HSCs) differentiate in successive steps of commitment and specification to mature erythrocytes. This differentiation process is controlled by transcription factors that establish stage- and cell type-specific gene expression. In this study, we demonstrate that FUSE binding protein 1 (FUBP1), a transcriptional regulator important for HSC self-renewal and survival, is regulated by T-cell acute lymphocytic leukaemia 1 (TAL1) in erythroid progenitor cells. TAL1 directly activates the FUBP1 promoter, leading to increased FUBP1 expression during erythroid differentiation. The binding of TAL1 to the FUBP1 promoter is highly dependent on an intact GATA sequence in a combined E-box/GATA motif. We found that FUBP1 expression is required for efficient erythropoiesis, as FUBP1-deficient progenitor cells were limited in their potential of erythroid differentiation. Thus, the finding of an interconnection between GATA1/TAL1 and FUBP1 reveals a molecular mechanism that is part of the switch from progenitor- to erythrocyte-specific gene expression. In summary, we identified a TAL1/FUBP1 transcriptional relationship, whose physiological function in haematopoiesis is connected to proper erythropoiesis.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Células Precursoras Eritroides/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ARN/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Células A549 , Proteínas de Unión al ADN/metabolismo , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células HEK293 , Células HL-60 , Humanos , Proteínas de Unión al ARN/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Regulación hacia ArribaRESUMEN
The transcriptional regulator far upstream element binding protein 1 (FUBP1) acts as an oncoprotein in solid tumor entities and plays a role in the maintenance of hematopoietic stem cells. However, its potential function in leukemia is unknown. In murine models of chronic (CML) and acute myeloid leukemia (AML) induced by BCR-ABL1 and MLL-AF9, respectively, knockdown of Fubp1 resulted in prolonged survival, decreased numbers of CML progenitor cells, decreased cell cycle activity and increased apoptosis. Knockdown of FUBP1 in CML and AML cell lines recapitulated these findings and revealed enhanced DNA damage compared to leukemia cells expressing wild type FUBP1 levels. FUBP1 was more highly expressed in human CML compared to normal bone marrow cells and its expression correlated with disease progression. In AML, higher FUBP1 expression in patient leukemia cells was observed with a trend toward correlation with shorter overall survival. Treatment of mice with AML with irinotecan, known to inhibit topoisomerase I and FUBP1, significantly prolonged survival alone or in combination with cytarabine. In summary, our data suggest that FUBP1 acts as cell cycle regulator and apoptosis inhibitor in leukemia. We demonstrated that FUBP1 might play a role in DNA repair, and its inhibition may improve outcome in leukemia patients.
Asunto(s)
Apoptosis , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide Aguda/patología , Proteínas de Unión al ARN/metabolismo , Animales , Trasplante de Médula Ósea , Ciclo Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Humanos , Irinotecán/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Inhibidores de Topoisomerasa I/farmacología , Células Tumorales CultivadasRESUMEN
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.
RESUMEN
The transcriptional regulator FUSE Binding Protein 1 (FUBP1) is overexpressed in more than 80% of all human hepatocellular carcinomas (HCCs) and other solid tumor entities including prostate and colorectal carcinoma. FUBP1 expression is required for HCC tumor cell expansion, and it functions as an important pro-proliferative and anti-apoptotic oncoprotein that binds to the single-stranded DNA sequence FUSE to regulate the transcription of a variety of target genes. In this study, we screened an FDA-approved drug library and discovered that the Topoisomerase I (TOP1) inhibitor camptothecin (CPT) and its derivative 7-ethyl-10-hydroxycamptothecin (SN-38), the active irinotecan metabolite that is used in the clinics in combination with other chemotherapeutics to treat carcinoma, inhibit FUBP1 activity. Both molecules prevent in vitro the binding of FUBP1 to its single-stranded target DNA FUSE, and they induce deregulation of FUBP1 target genes in HCC cells. Our results suggest the interference with the FUBP1/FUSE interaction as a further molecular mechanism that, in addition to the inactivation of TOP1, may contribute to the therapeutic potential of CPT/SN-38. Targeting of FUBP1 in HCC therapy with SN-38/irinotecan could be a particularly interesting option because of the high FUBP1 levels in HCC cells and their dependency on FUBP1 expression.